Endothelial cell differentiation and establishment of blood vessel identity is essential for vascular function in both normal and disease settings. For example, in the developing embryo differentiation of arterial and venous endothelial cells is essential for proper vessel patterning and circulatory function. Similarly, perturbation of blood vessel identity in a number of congenital diseases can lead to vascular anomalies, such as arteriovenous malformations. Thus, understanding the molecular basis of normal endothelial differentiation would give us insights onto pathological blood vessel formation and facilitate the engineering of distinct blood vessel types. While transcriptional hierarchies controlling cellular differentiation have been extensively characterized in other tissues, much less is known about such programs in endothelial cells. The studies proposed in this application will begin to address the control of artery and vein differentiation at the transcriptional level in endothelial cells. In particular, we will identify cis regulatory elements in the human genome that are responsible for arterial and venous endothelial-specific gene expression. This will be accomplished through the genome-wide identification of binding sites for general transcriptional regulatory proteins known to reliably mark enhancer and repressor elements. Subsequent computational analyses will allow us to identify common cis regulatory sequences that correlate with artery or vein specific gene expression. These will shed possible insight onto upstream transcriptional regulators. To functionally validate the activity of putative artery and vein cis elements, we will perform in vivo reporter assays on human elements using the zebra fish as a model system. The transparency and external development of the zebra fish embryo, coupled with its rapid development will allow us to comprehensively determine the activity of a large number of cis elements for numerous arteries and vein restricted genes. Together, these studies will allow us to map the transcriptional regulatory inputs that contribute to arterial and venous endothelial differentiation and blood vessel identity. PUBLIC HEALTH RELEVANCE: Blood vessels have different functions and appearances (e.g. artery versus vein) depending on their anatomical location. These differences are apparent in early embryos and may play a role in disease settings. However, little is known about how these differences arise. In this proposal, we will focus on uncovering the signals that govern artery and vein identity.